Benjamin Fisch

Morphological study of fully and partially isolated early human follicles

OBJECTIVE To compare the development of fully and partially isolated human follicles by using various culture systems. DESIGN Human ovarian material was incubated with collagenase and deoxyribonuclease. Fully and partially isolated follicles (30-50 microm) were dissected and studied under light and electron microscopy. The follicles were then cultured on and within various matrices. Fully isolated follicles were also cocultured with stromal cells. SETTING Rabin Medical Center, a major care and referral center. PATIENT(S) Women undergoing laparoscopy. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Microscopy studies, follicular measurements. RESULT(S) Electron microscopy studies revealed an excess of lipid droplets in the granulosa cells of freshly isolated follicles. An increase in follicular size and granulosa cell number was observed only in the fully isolated follicles cultured within collagen gels for 24 hours. Most of the partially isolated follicles detached from the collagen gels. When cultured on collagen, extracellular matrix, and poly-L-lysine, both the fully and the partially isolated follicles deteriorated within the first 24 hours; coculture with stromal cells had no beneficial effect. CONCLUSION(S) The excess in lipid droplets in granulosa cells of isolated follicles might suggest that the isolation process does not yield completely healthy follicles. However, despite this finding, our studies show that fully isolated follicles, but not partially isolated follicles, can grow within, but not on, a culture matrix.

Assisted hatching by partial zona dissection of human pre-embryos in patients with recurrent implantation failure after in vitro fertilization*

OBJECTIVE To examine the potential of the partial zona dissection technique to promote successful implantation by assisting embryo hatching after IVF. DESIGN The study and the control group included 72 and 82 patients, respectively, each had undergone at least three failed IVF-ET attempts. Assisted hatching was performed on four- to six-cell stage embryos by creating a slit in the zona pellucida using the partial zona dissection technique. After 90 minutes incubation (5% CO2, 37 degrees C), the embryos were transferred to the uterus. SETTING Infertility and IVF Unit of an academic tertiary referral medical center. RESULTS In the assisted hatching group, 230 micromanipulated embryos were replaced (3 or 4 treated embryos per patient) compared with 295 nonmanipulated embryos in the control group. Clinical pregnancy rates (PRs) were similar in the assisted hatching and control groups (n = 15; 20.8% and n = 12; 14.6%, respectively). However, the contribution of assisted hatching by partial zona dissection to successful implantation was related to the patientss age: patients older than 38 years showed a markedly higher PR after assisted hatching: 23.9% in the study group compared with only 7% of the controls. CONCLUSIONS These results demonstrate that assisted hatching by partial zona dissection is a quick and efficient method that does not induce any visible damage to the embryos replaced. In a selected group of patients (aged over 38 years, who have failed to conceive in at least three previous IVF attempts) it significantly increases the chances for pregnancy after ET.

The relationship between in vitro fertilization and naturally occurring antibodies: evidence for increased production of antiphospholipid autoantibodies*

OBJECTIVE Assessment of possible effects of ovarian stimulation during in vitro fertilization (IVF) treatment cycles on circulating levels of antiphospholipid and antinuclear autoantibodies. DESIGN The study was performed prospectively. Sera were obtained at three time points along IVF treatment cycle. Levels of autoantibodies directed against nuclear components, mitochondrial antigens, and phospholipids were determined using enzyme-linked immunosorbent assay. PATIENTS Thirty-five patients, who underwent at least one previous IVF attempt, and 36 age- and sex-matched controls were analyzed. All participants were randomly selected. RESULTS The mean levels of antiphospholipid (but not antinuclear) autoantibodies in sera from IVF-treated patients were found to be significantly higher than the corresponding values of the control group (for immunoglobulin [Ig]M isotype: anticardiolipin, antiphosphatidyl L-serine; for IgG isotype: anticardiolipin, antiphosphatidyl L-serine, and antiphosphatidylcholine; P less than 0.0001, assessed by Mann-Whitney test). The autoantibody levels remained more or less constant at different time points along the treatment cycle. No correlation with age and number of previous IVF cycles was demonstrated. CONCLUSIONS Serum levels of antiphospholipid (but not antinuclear) autoantibodies increase after IVF treatment. Based on these preliminary data, it is not yet possible to estimate if the observed changes in autoantibody levels might have any future clinical influence on infertile patients undergoing IVF treatment.

Occasional involvement of the ovary in Ewing sarcoma

BACKGROUND Ewing sarcoma (EWS) is a highly metastatic malignancy in young patients. Ovarian cryopreservation is often an option for fertility preservation in cancer patients of reproductive age, specifically in minors. Thus, the possibility of ovarian involvement in EWS needs to be elucidated. METHODS Eight patients aged 13-20 years with EWS participated in the study. Ovarian samples were fixed and prepared for light microscopy, and frozen in liquid nitrogen for RNA extraction followed by RT-PCR. Histological studies, including immunostaining for the adhesion receptor CD99, were used to detect histopathological features. Sensitive molecular methods were used to detect translocations causing the formation of tumor-specific EWS-Friend leukemia virus integration site 1 fusion gene (EWS-FLI1). RESULTS In seven patients, there was no evidence of EWS in the ovaries from pathological/molecular studies. However, in one patient, the RT-PCR showed the EWS translocation, although there was no pathological evidence. CONCLUSIONS Ovarian involvement is possible in EWS. Therefore, in patients with EWS ovarian tissue should be examined for traces of malignancy at both the pathological and molecular levels prior to the grafting of cryopreserved tissue in order to minimize the risk of reseeding the cancer.

Possible improvements in human ovarian grafting by various host and graft treatments

BACKGROUND Anticancer treatment poses a high risk of ovarian failure. In many cases cryopreservation of ovarian tissue is the only option for fertility preservation. Although autologous transplantation of cryopreserved-thawed ovarian tissue has resulted in live births, slow graft revascularization and ischemia after transplantation leads to substantial follicular loss. Therefore, methods to improve and hasten graft vascularization are needed. The aim of the study was to examine the benefits of host and graft treatments with melatonin, hyaluronan (HA), vascular endothelial growth factor A (VEGF-A) and vitamin E with regard to the outcome of human ovarian tissue grafting. METHODS Five young cancer patients who underwent laparoscopic ovarian surgery for fertility preservation donated ovarian tissue. Thawed ovarian samples were transplanted into immunodeficient mice divided into seven groups: (A) no treatment; (B) host treatment with melatonin before and after grafting; (C) graft incubation with HA-rich biological glue before transplantation; (D) host as in (B), graft as in (C); (E) host as in (B), graft incubation with VEGF-A and vitamin E; (F) graft as in (C) combined with VEGF-A and vitamin E; (G) host as in (B), graft as in (F). Graft survival was assessed by follicle counts, apoptosis assay and immunohistochemical staining for proliferating cell nuclear antigen and VEGF-A expression. RESULTS Only grafts implanted in melatonin-treated hosts and grafts incubated with HA-rich biological glue retained their original size. Apoptosis was significantly lower after host treatment with melatonin and graft incubation with HA-rich biological glue plus VEGF-A and vitamin E than in untreated grafts; apoptosis was specifically low in Group G. There were significantly more atretic follicles in the untreated group than in most treated groups. CONCLUSIONS The findings suggest that host treatment with melatonin or graft incubation with HA-rich biological glue, especially when combined with VEGF-A and vitamin E improves graft survival. This protocol can be applied and holds promise in ovarian autotransplantation for fertility restoration.

The relationship between sperm ultrastructural features and fertilizing capacity in vitro

OBJECTIVE To evaluate the relationship between ultramorphological features of the human sperm and its fertilizing capacity in vitro. DESIGN The study was performed retrospectively. Ultrastructural features were assessed using scanning and transmission electron microscopes in sperm samples of individuals who underwent an in vitro fertilization (IVF) treatment cycle no more than 6 months before the study. SETTING Institutional clinical care. PATIENTS Fifty-six infertile couples in whom mechanical infertility was diagnosed in the female partner. Patients were categorized as fertilizing when fertilization of at least 30% of the oocytes occurred (n = 27) and nonfertilizing when none of the oocytes fertilized in at least two consecutive IVF treatment cycles (n = 29). RESULTS The two groups differed significantly only in ultramorphological parameters of the sperm head and acrosome (head, F(8,36) = 2.8, P less than 0.02; acrosome, F(4,40) = 2.8, P less than 0.04), and especially in the following malformation patterns: hyperelongated head, acrosome deficiency, and acrosome damage. The suggested score based on these findings was able to predict 90% and 76% of the cases with and without fertilizing potential, respectively. CONCLUSION The ultrastructural morphology of the sperm head components is a key parameter for assessing the sperm fertilizing capacity in vitro.

Growth differentiating factor 9 (GDF9) and bone morphogenetic protein 15 both activate development of human primordial follicles in vitro, with seemingly more beneficial effects of GDF9.

CONTEXT The signals initiating growth of primordial follicles are unknown. Bone morphogenetic protein 15 (BMP15) and growth differentiating factor 9 (GDF9) are promising candidates. OBJECTIVE The objective of the study was to evaluate for the first time the effects of human recombinant BMP15 and human recombinant GDF9 on the in vitro development of human primordial follicles. DESIGN AND SETTING This was a controlled culture study performed in a major tertiary university-affiliated medical center. MATERIALS Materials included ovarian tissue from 17 girls/women and three aborted human fetuses. INTERVENTION There were no interventions. MAIN OUTCOME MEASURE Histological and immunohistochemical (proliferating cell nuclear antigen, BMP15, and GDF9) studies and an endocrine assay of 17β-estradiol were conducted. RESULTS In the samples from girls/women, the number of developing follicles was greater with GDF9 or BMP15 alone than with no BMP15 or GDF9. Higher 17β-estradiol secretion was noted after treatment with GDF9 than with BMP15 or with GDF9+anti-GDF9. The number of atretic follicles was greater with BMP15 than with GDF9. Proliferating cell nuclear antigen expression was greater with the higher dose of both growth factors than the lower dose. Expression of BMP15 and GDF9 was identified in samples cultured without BMP15 or GDF9. Results for the fetal follicles yielded no distinguishable pattern. CONCLUSIONS Although both BMP15 and GDF9 promoted activation of human primordial follicles from girls/women (but not human fetuses) in a dose-dependent manner, GDF9 seems more beneficial.

Preliminary studies on apoptosis in human fetal ovaries

OBJECTIVE To evaluate if apoptosis occurs in human germ cells between 19 and 33 gestational weeks (GW). DESIGN Human fetal ovaries were obtained from aborted fetuses aged 19-33 GW. SETTING Rabin Medical Center, a major tertiary care and referral center. PATIENT(S) Twenty-seven women undergoing pregnancy termination. The abortions were mostly because of fetal anatomical or chromosomal abnormalities. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Microscopy studies, terminal deoxynucleotidyl transferase (TdT) assay (TUNEL), and immunocytochemistry for B-cell lymphoma/leukemia-2 (bcl-2). RESULT(S) TUNEL assay revealed a slight increase in apoptotic oocytes in fetuses from 23 GW, with a peak at 27 GW. Overexpression of bcl-2 was detected in all ovarian components, regardless of fetal age. CONCLUSION(S) There seems to be a slight increase in apoptosis in oocytes from 23 GW with a peak at 27 GW. However, it is very unlikely that these low apoptotic rates could be the cause of the extensive germ cell loss throughout human pregnancy. The overexpression of bcl-2 possibly suggests either that this gene is necessary to overcome extensive apoptotic activity or that it is responsible for the low apoptosis rates. However, these results should be considered with caution, since the ovaries were mostly from abnormal fetuses after feticide.

High serum oestradiol concentrations in IVF cycles increase the risk of pregnancy complications related to abnormal placentation

The study was designed to evaluate the isolated effect of high serum oestradiol concentration on human chorionic gonadotrophin (HCG) day in IVF cycles on endometrial receptivity and placentation. A retrospective cohort included all women attending the IVF unit in 2006 and 2007, with the best prognosis to achieve pregnancy: age (<38 years), less than three IVF cycles, transfer of two highest grade embryos and no evidence of factors known to impair implantation or that are associated with increased risk of pregnancy complications. The total included 280 patients were categorized into three groups according to their serum oestradiol concentration on HCG day: group 1, oestradiol <5000 pmol/l, group 2, oestradiol in the range 5000-10,000 pmol/l and group 3, oestradiol in the range of 10,000-15,000 pmol/l. No significant differences were found between the groups in implantation, pregnancy and abortion rates. The high oestradiol group was characterized by high rate (20.8%) of pregnancy complications related to abnormal placentation--fetal growth restriction, pregnancy-induced hypertension and abnormal implantation of the placenta. Hence, the decision to perform embryo transfer in high-responder patients should take into consideration both possible risks of ovarian hyperstimulation syndrome and pregnancy complications related to abnormal placentation.

Improving posttransplantation survival of human ovarian tissue by treating the host and graft.

OBJECTIVE To improve posttransplantation survival of frozen-thawed human ovarian tissue in immunodeficient mice. DESIGN Histologic study of transplanted human ovaries after treating the host and graft. SETTING Infertility unit, university-affiliated tertiary medical center. PATIENT(S) Ovarian tissue from six girls/women (aged 5-23 years) who had undergone ovarian laparoscopy for fertility preservation. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Thawed ovarian samples were transplanted into the back muscle of immunodeficient mice divided into four groups: A) no treatment; B) host treatment with vitamin E and gonadotropins before and after grafting; C) graft incubation with vascular endothelial growth factor A (VEGF-A) and vitamin E before transplantation; and D) host as in B, graft as in C. Ungrafted thawed samples served as control. Assessment of graft survival was conducted by follicle counts, apoptosis evaluation, immunohistochemical stainings for proliferating cell nuclear antigen (PCNA) and VEGF-A expression. RESULT(S) Only grafts incubated before transplantation (groups C and D) retained their original size. Follicle number was low in all grafts. PCNA expression was found in most grafts. Apoptosis was significantly lower in the untreated and treated grafts transplanted into treated hosts (groups B and D) than in ungrafted-thawed samples and group A grafts. All grafted groups had significantly higher expression of VEGF-A than ungrafted-thawed samples. CONCLUSION(S) Survival of transplanted human ovarian tissue may be improved by treatment of the host and graft. Further studies to evaluate treatments with a potential benefit in human ovarian autotransplantation are needed.